Process for the isolation and purification of epothilones

ABSTRACT

The invention relates to a desorption process for epothilones, especially epothilone A and/or epothilone B, from resins and new production, work-up or purification processes or manufacturing methods for epothilones comprising said desorption process, as well as the use of certain solvents for the desorption of epothilones from resins.

The invention relates to a new desorption process for epothilones,especially epothilone A and/or epothilone B, from resins and newproduction, work-up or purification processes or manufacturing methodsfor epothilones comprising said desorption process, as well as the useof certain solvents for the desorption of epothilones from resins.

BACKGROUND OF THE INVENTION

Epothilones A and B represent a new class of microtubuli-stabilisingcytotoxic active ingredients (see Gerth, K. et al., J. Antibiot. 49,560-3 (1966)) of the formulae:

wherein R signifies hydrogen (epothilone A) or methyl (epothilone B).

Since the description of these epothilones (see WO 93/10121), severalmethods have been disclosed for the synthesis and manufacture both ofthe epothilones and mainly of numerous epothilone derivatives(collectively called “epothilones” hereinafter), for example thosedisclosed in WO 99/03848, WO 00/49020, WO 00/49021, WO 00/47584, WO00/00485, WO 00/23452, WO 99/03848, WO 00/49019, WO 99/07692, WO98/22461, WO 99/65913, WO 98/38192, WO 00/50423, WO 00/22139, WO99/58534, WO 97/19086, WO 98/25929, WO 99/67252, WO 99/67253, WO00/31247, WO 99/42602, WO 99/28324, WO 00/50423, WO 00/39276, WO99/27890, WO 99/54319, WO 99/54318, WO 99/02514, WO 99/59985, WO00/37473, WO 98/08849, U.S. Pat. No. 6,043,372, U.S. Pat. No. 5,969,145,WO 99/40047, WO 99/01124, and WO 99/43653. In addition to epothilone Aand epothilone B, especially the epothilones D and E, described in WO97/19086 and WO 98/22461, and the epothilones E and F, described in WO98/22461, as well as the epothilones described in WO 99/02514 showinteresting properties.

As an example for the therapeutic use, International Application WO99/43320 describes a number of ways of administration of epothilones asagents against proliferative diseases, especially tumor diseases, that,due to their similar mechanism, act in a way comparable to that ofTaxol®, a well-known and marketed anticancer agent. WO 99/39694discloses some specific formulations of the epothilones, especially Aand B.

The epothilones, especially epothilone A and most preferably epothiloneB, offer a number of advantages in comparison to established treatments,especially also in cases where tumors have become refractory to thetreatment with Taxol. Therefore, methods for their synthesis in largeramounts are urgently required in order to meet anticipated demands.

The most efficient production processes so far at least comprise somebiosynthetic steps and isolation of epothilones from culture media orthe like.

Originally, the extraction of natural substances by means ofmyxobacteria, especially the epothilones from the cell strain SorangiumCellulosum Soce90 (deposited under no. 6773 at the German Collection ofMicroorganisms, see WO 93/10121) was described in literature. In orderto obtain a satisfactory concentration of the natural substances,especially the epothilones, previously an adsorbate resin based onpolystyrene was always added to the culture medium for absorption to themedium, for example Amberlite XAD-1180 (Rohm & Haas, Frankfurt,Germany).

However, the disadvantage of this process is that, on a large scale, itleads to an abundance of problems. Valves are impaired by the globulesof resin, pipes can block, and apparatus may be subject to greater weardue to mechanical friction. The globules of resin are porous andtherefore have a large inner surface area (about 825 m²/gram resin).Sterilisation becomes a problem, as air enclosed in the resin is notautoclaved. Thus, the process cannot be practicably carried out on alarge scale using resin addition during cultivation of themicroorganisms that produce epothilones.

Therefore an advanced process for the production of epothilones,especially of epothilones A and B, was found and described in WO99/42602. That method comprises complexing of epothilones from culturemedia of epothilone-producing microorganisms, said media comprisingcyclodextrines or other complex-forming agents, mixing of the cell-freeculture medium (e.g. filtrate or centrifugate of said culture medium)with a synthetic resin, for example a resin based onstyrene/divinylbenzene copolymers as matrix, such as Amberlite XAD-16(Rohm & Haas Germany GmbH, Frankfurt, Germany) or Diaion HP-20(Resindion S.R.L., Mitsubishi Chemical Co., Milan, Italy) in order toabsorb the epothilones and desorption, especially with an alcohol, mostpreferably isopropanol. This is followed by addition of water to thealcohol phase, removal of the solvent phase (preferably by evaporation),phase separation of the resulting residue in the presence of an ester,especially ethyl acetate or isopropyl acetate, usually molecularfiltration (gel chromatography) of the dried ester phase, separation ofthe resulting epothilone mixture by reverse phase HPLC (preferably byelution with a mixture of nitrile/water, e.g. acetonitrile/water), andoptionally further purification by phase separation in the presence of awater/ether mixture, preferably subsequent adsorption chromatography onsilica gel in order to achieve further removal of impurities, andcrystallisation/recrystallisation.

Though a useful progress and appropriate for industrial scaleproduction, this method still suffers from certain disadvantages.

For example, in order to obtain sufficient purity, it is advisable tomake use of either the molecular filtration step or the silica geladsorption chromatography step or both. More difficulties come from thephase separation in the presence of an ester, such as ethyl acetate,which (especially due to the long time for phase separation of thewater/ester phase in large industrial scale) is very time-consuming, aswell as the subsequent evaporation, which in addition is difficult tohandle in view of foaming and sputtering.

It is thus a problem to be solved by the present invention to avoid asmany of the above difficulties as possible and find new and advantageousways for the isolation of epothilones, especially epothilones A and B,after their adsorption to a resin.

GENERAL DESCRIPTION OF THE INVENTION

Very surprisingly, it has now been found that by simple replacement ofthe alcohols used as desorption solvent with certain other solvents(named weakly polar or apolar solvents as specified below) provides areal breakthrough that can be achieved in order to obtain a solution tothe problems mentioned above, leading to additional advantages such asimproved desorption and higher final yield. Among the additionaladvantages are (i) higher selectivity of desorption; (ii) higher amountof desorbed epothilones, indicating more complete desorption; (iii) nore-extraction with ester (e.g. ethyl acetate) with the highlyproblematic water/ester phase separation and no subsequent etherevaporation required; (iv) no molecular filtration and usually noadsorption chromatography are required; (v) the time required fordesorption is diminished considerably; (vi) fewer process steps, (vii)diminished contamination risk (important with the highly toxicepothilones), and/or (viii) better and safer handling (depending on thesolvent used for desorption=extraction); and (ix) also depending on thesolvent unexpectedly even lower amounts of by-products or impuritieswith similar polarity as the epothilones, especially as epothilone B,need to be removed after reversed phase chromatography. These andfurther advantages can be deduced from the details given in thesubsequent Detailed Description of the Invention:

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to a process for desorbingepothilones, especially epothilone A and/or B, in particular epothiloneB, from a resin, especially a synthetic resin, with a weakly polar orapolar solvent.

A further aspect relates to a process for working up (or purifying)epothilones, especially after their production in a standard medium forchemical synthesis or preferably a culture medium which comprisesmicroorganisms, especially myxobacteria, in particular of the genusSorangium, which are suitable for producing epothilones, especiallyepothilone A and/or B, and a complex-forming component, said processcomprising the use of a weakly polar or apolar solvent for thedesorption of said epothilones from a resin.

A third aspect of the invention relates to the use of a weakly polar orapolar solvent for the desorption of epothilones, especially epothiloneA and/or B, in particular epothilone B, from a resin, especially asynthetic resin.

The general terms used hereinabove and hereinbelow preferably have themeanings given hereinbelow:

The term “epothilones” preferably relates to any epothilone orepothilone derivative mentioned in the patent applications WO 99/03848,WO 00/49020, WO 00/49021, WO 00/47584, WO 00/00485, WO 00/23452, WO99/03848, WO 00/49019, WO 99/07692, WO 98/22461, WO 99/65913, WO98/38192, WO 00/50423, WO 00/22139, WO 99/58534, WO 97/19086, WO98/25929, WO 99/67252, WO 99/67253, WO 00/31247, WO 99/42602, WO99/28324, WO 00/50423, WO 00/39276, WO 99/27890, WO 99/54319, WO99/54318, WO 99/02514, WO 99/59985, WO 00/37473, WO 98/08849, U.S. Pat.No. 6,043,372, U.S. Pat. No. 5,969,145, WO 99/40047, WO 99/01124, and/orWO 99/43653, more preferably to epothilone A and especially epothiloneB, but in a broader aspect of the invention also to the epothilones Dand E, described in WO 97/19086 and WO 98/22461, the epothilones E andF, described in WO 98/22461, or the epothilones described in WO99/02514. All these documents, especially with regard to the epothilonederivatives and the preferred epothilone derivatives mentioned therein,are herewith enclosed by reference.

A weakly polar or apolar solvent preferably has the followingcharacteristics:

Preferred is a solvent that, in the eluotropic row determined accordingto Snyder et al., J. Chromatogr. Sci. 16, 223 (1978), shows thefollowing characteristics, where x_(e) is the proton acceptor parameter(indicator of the tendency to form hydrogen bonds as hydrogen acceptor),X_(d) is the proton donor parameter (indicator of the tendency to formhydrogen bonds as hydrogen donor) and x_(n) is the dipole parameter(indicating the dipole character), with the proviso thatx_(e)+x_(d)+x_(n)=1, or a mixture of such solvents:

-   -   x_(e)=0.20-0.40; x_(d)=0.15-0.36; and x_(n)=0.38-0.60;        preferably    -   x_(e)=0.22-0.32; x_(d)=0.17-0.34; and x_(n)=0.39-0.54.

Very preferred among the weakly polar or apolar solvents are loweralkyl-lower alkyl ketones or cyclic ketones, such as acetone, methylethyl ketone, 2-pentanone, methyl-isobutyl-ketone or cyclohexanone, morepreferably ethers, especially cyclic ethers, more especiallytetrahydrofuran or dioxan; still more preferably aliphatic halogencompounds, especially lower alkyl halogenides, especially methylenedichloride (=methylenechloride) or ethylene dichloride; or mostespecially aromatic solvents, especially naphthalene or (preferably)benzene or naphthaline or (preferably) benzene substituted by one ormore, preferably one to three moieties selected from the groupconsisting of lower alkyl, especially methyl, ethyl or isopropyl, loweralkoxy, especially methoxy or ethoxy, halogen, especially fluoro,chloro, bromo or iodo, nitro and lower alkoxy-lower alkyl, especiallyethoxymethyl; especially toluene, ethyl-benzene, xylene, especially o-,m- or p-xylene, mesitylene, pseudo-cumene, hemellitene, cumene,isopropyl-toluene, phenyl halogenides, especially fluorobenzene,chlorobenzene, bromobenzene or iodobenzene, lower alkoxy benzenes,especially ethoxybenzene or methoxybenzene, or lower alkoxy-lower alkylbenzenes, especially ethoxymethyl benzene (benzyl ether); or any mixtureof two or more, for example 2 to 4 of these solvents; most preferred arelower alkyl-benzenes, especially ethyl-benzene, xylene, especially o-,m- or p-xylene, mesitylene, pseudo-cumene, hemellitene, cumene,isopropyl-toluene, and most especially toluene.

The term “weakly polar or apolar solvent” also includes mixtures of twoor more of the solvents described hereinbefore and hereinafter, e.g. of2 to 4 such solvents.

The prefix “lower” always indicates that the correspondingly namedradical contains preferably up to a maximum of 7 carbon atoms, inparticular up to 4 carbon atoms, and is branched or unbranched. Loweralkyl may be for example unbranched or branched once or more, and ise.g. methyl, ethyl, propyl such as isopropyl or n-propyl, butyl such asisobutyl, sec-butyl, tert-butyl or n-butyl, or also pentyl such as amylor n-pentyl.

Halogen is preferably iodo, bromo, chloro or fluoro.

A hydrocarbon preferably is an organic compound having 4 to 32, morepreferably 4 to 20, still more preferably 6 to 16 carbon atoms and maybe aliphatic, e.g. a linear, branched or cyclic saturated alkane, e.g;cyclohexane, a linear, branched or cyclic (non-aromatic) organiccompound with one or more double and/or triple bonds, or an aromatichydrocarbon, the latter being unsubstituted or substituted by one ormore, e.g. up to three, substituents selected from the group consistingof lower alkyl, especially methyl, ethyl or isopropyl, lower alkoxy,especially methoxy or ethoxy, halogen, especially fluoro, chloro, bromoor iodo, nitro and lower alkoxy-lower alkyl, especially ethoxymethyl;preferably by one to three lower alkyl moieties.

An alcohol is preferably a hydroxy-lower alkane, especially methanol,ethanol or n- or iso-propanol.

A resin is especially a synthetic resin, preferably a resin based onstyrene and divinylbenzene copolymers, more preferably Amberlite XAD-4or preferably Amberlite XAD-16 [Rohm & Haas Germany GmbH, Frankfurt] orDiaion HP-20 [Resindion S.R.L., Mitsubishi Chemical Co., Milan]. It goeswithout saying that a resin from which an epothilone or epothilones areto be desorbed in accordance with the invention has epothilonesnon-covalently in contact with it (e.g. reversibly bound to it oradsorbed to it). In other terms, where the term “resin” is used, it isintended to mean “resin having one or more epothilones in contact withit, especially reversibly bound or adsorbed to it”.

PREFERRED ASPECTS OF THE INVENTION

In a preferred aspect of the invention, the invention relates to aprocess comprising a desorption from a resin with a weakly polar orapolar solvent as described above or below, comprising any furtherpurification steps desirable or required to come to the respectiveepothilones, especially epothilone A and most especially epothilone B,in pure form, preferably those described as preferable in the presentinvention.

Working up of the epothilones is preceded by the reaction or processleading to a reaction product comprising epothilones to be worked upwhich are isolated (i) either from chemical reaction mixtures aftersolution in an appropriate polar aqueous medium, or (ii) more preferablyfrom the supernatant (for example a cyclodextrine containing one asdescribed in WO 99/42602) of a culture medium with microorganismsproducing the epothilones by separating a culture into the liquid phase(e.g. centrifugate or filtrate) and solid phase (cells), especially bymeans of filtration or centrifugation (tubular centrifuge or separator).

This pretreatment is then preferably continued by directly mixing thesolution ((i)) or liquid phase ((ii)) with a resin, especially asynthetic resin, especially a resin based on styrene and divinylbenzenecopolymers as matrix (hereinafter referred to also simply as polystyreneresin), such as Amberlite XAD-16 or Dialon HP-20 (preferably in a ratioof centrifugate: resin volume of ca. 10:1 to 100:1, preferably about50:1). After a period of contact of preferably 0.25 to 50 hours,especially 0.8 to 22 hours, the resin is separated, for example byfiltration, sedimentation or centrifugation. If required, afteradsorption the resin is washed with a strongly polar solvent, preferablywith water.

Then the preferred process of working up according to the presentinvention starts: Desorption of the epothilones (as such forming a verypreferred aspect of the invention) is effected with a weakly polar orapolar solvent, in particular with lower alkyl halogenides, especiallymethylene dichloride or ethylene dichloride, or more preferably aromaticsolvents, especially naphthalene or (preferably) benzene, or naphthalineor (preferably) benzene substituted by one or more, preferably one tothree moieties selected from the group consisting of lower alkyl,especially methyl, ethyl or isopropyl, lower alkoxy, especially methoxyor ethoxy, halogen, especially fluoro, chloro, bromo or iodo, nitro andlower alkoxy-lower alkyl, especially ethoxymethyl; most preferablytoluene. The step of extraction may be repeated one or more times,preferably 0 to 3 times, especially once, in order to obtain morecomplete desorption where necessary or desired.

The working up of the (in case of repeated extraction combined)solutions of desorbed epothilones thus obtainable preferably continueswith the removal of the solvent used for desorption from the resultingsolutions by evaporation (distillation), preferably by means ofconcentration in a reactor and subsequently in a rotary evaporator undervacuum.

Subsequently, further processing takes place using the following steps,where the purification step by means of reversed-phase chromatographywith elution with a nitrile is compulsory, while the other steps areoptional:

crystallization of the epothilone(s) after desorption; especially, forthe crystallization of epothilone B, a mixture of an alcohol and ahydrocarbon, especially of a lower alkanol and a cyclic aliphatichydrocarbon with 3 to 10 ring atoms, most especially of methanol andcyclohexane (preferably in a v/v ratio of 1:10 to 10:1, especially of1:3 to 3:1, is added. Addition of water leads to fast phase separation,and the alcohol phase is evaporated to dryness, e.g. by means of arotary evaporator under vacuum. The resulting extract which comprisesthe epothilone B is afterwards crystallized from an appropriate solventmixture, especially from an alcohol/cyclic aliphatic hydrocarbonmixture, most preferably isopropanol/cyclohexane, preferably in a v/vratio of 1:10 to 10:1, more preferably 1:6 to 6:1, most preferably 1:6to 1:4;

(obligatory) separation of the epothilones by reversed-phasechromatography after being taken up in a suitable solvent, especially amixture of a nitrile and water, preferably acetonitrile/water, in apreferred v/v ratio of 1:10 to 10:1, especially 1:3 to 1:1, and elutionwith a mixture of nitrile and water, preferably characterised in thatthe chromatography is carried out on column of a reversed phasematerial, which is charged with hydrocarbon chains, such as hydrocarbonchains containing 18 carbon atoms, especially an RP-18 material, and aneluant comprising a nitrile, especially a lower alkylnitrile, inparticular acetonitrile, is used, in particular a mixture ofnitrile/water is used, especially a mixture of acetonitrile/water,preferably in a ratio of nitrile to water of about 1:99 to 99:1,primarily between 1:9 and 9:1, e.g. between 2:8 and 7:3, e.g. 3:7 or4:6; and removal of the nitrile from the collected epothilone(especially epothilone A or most especially epothilone B) comprisingfractions by evaporation (distillation); if desired, the remaining waterwith the epothilone is then extracted with an ester, especially a loweralkyl-lower alkanoate, preferably isopropyl acetate, with subsequentevaporation (preferably first in a reactor, then in a rotary evaporatorunder vacuum) of the epothilone containing ester phase to dryness; (ifrequired, the starting epothilone solution may be split and separated inmore than one reverse phase separate runs;)

only if required (e.g. as an alternative to the crystallization afterdesorption) adsorption chromatography, in particular by adding to acolumn of silica gel and eluting with an appropriate solvent or solventmixture, especially a mixture of ester/hydrocarbon, for example loweralkyl alkanoate/C₄-C₁₀-alkane, especially ethyl or isopropylacetate/n-hexane, in which the ratio between the ester and hydrocarbonis preferably in the range 99:1 to 1:99, preferably 10:1 to 1:10, forexample 4:1;

and finally recrystallisation, e.g. from appropriate solvents or solventmixtures, for example consisting of esters, ester/hydrocarbon mixturesor alcohols, especially ethyl or isopropyl acetate:toluene 1:10 to 10:1,preferably 2:3 (epothilone A) or methanol or ethyl acetate (epothiloneB);

in which process, if necessary and/or desired, between each step beingemployed, the resulting solutions or suspensions are concentrated,and/or liquid and solid components are separated from one another, inparticular by sedimenting, filtering or centrifuging ofsolutions/suspensions. The more precise definitions mentioned above andbelow can be preferably used in the above individual steps.

A preferred aspect of the invention relates also to a process for theisolation of epothilones adsorbed to a synthetic resin, especiallyepothilone A or most especially epothilone B, which process comprises

-   -   (i) the desorption of the epothilones from said synthetic resin        with a weakly polar or apolar solvent, in particular with lower        alkyl halogenides, especially methylene dichloride or ethylene        dichloride, or more preferably aromatic solvents, especially        naphthalene or (preferably) benzene, or naphthaline or        (preferably) benzene substituted by one or more, preferably one        to three moieties selected from the group consisting of lower        alkyl, especially methyl, ethyl or isopropyl, lower alkoxy,        especially methoxy or ethoxy, halogen, especially fluoro,        chloro, bromo or iodo, nitro and lower alkoxy-lower alkyl,        especially ethoxymethyl; most preferably toluene; or in a        broader aspect of the invention a mixture of two or more such        solvents; and    -   (ii) separation of the epothilones by reversed-phase        chromatography after being taken up in a suitable solvent,        especially a mixture of a nitrite and water, preferably        acetonitrile/water, in a preferred v/v ratio of 1:10 to 10:1,        especially 1:3 to 1:1, and elution with a mixture of nitrite and        water, preferably characterised in that the chromatography is        carried out on column of a reversed phase material, especially        an RP-18 material, which is charged with hydrocarbon chains,        such as hydrocarbon chains containing 18 carbon atoms, and an        eluant comprising a nitrile, especially a lower alkylnitrile, in        particular acetonitrile, is used, in particular a mixture of        nitrile/water is used, especially a mixture of        acetonitrile/water, preferably in a ratio of nitrile to water of        about 1:99 to 99:1, primarily between 1:9 and 9:1, e.g. between        2:8 and 7:3, e.g. 3:7 or 4:6.

In a further preferred aspect of the invention, the process in the lastparagraph starting with step (i) and comprising step (ii) furtherimplies any further purification steps desirable or required to come tothe respective epothilones, especially epothilone A and most especiallyepothilone B, in pure form, preferably those described as preferable inthe present invention.

Preparation for working up is preferably carried out as follows:Adsorption of the epothilones, especially from chemical reactionmixtures or more preferably from the supernatant of cultures ofmicroorganisms, can be achieved as described in WO 99/42602 or inanalogy thereto; briefly, the epothilones are found in the centrifugate,which is then directly mixed with a synthetic resin, especially astyrene/divnylbenzene copolymer resin, such as Amberlite XAD-16 orDiaion HP-20 (preferably in a ratio of centrifugate: resin volume of ca.10:1 to 100:1, preferably about 50:1) and stirred in an agitator. Inthis step, the epothilones are transferred to the resin. After a periodsufficient for adsorption, e.g. period of contact of ca. 0.2 to 10 h,the resin is separated by centrifugation or filtration. Adsorption ofthe epothilones onto the resin may also be effected in a chromatographycolumn, by placing the resin in the column and running the centrifugateover the resin. After adsorption, the resin is washed with water.

The preferred processes according to the invention then start andproceed as follows: Desorption of the epothilones from the resin ispreferably effected with a weakly polar or apolar solvent according tothe invention, especially one described as preferred above or below,especially methylenechloride or most especially toluene. The solvent isthen removed as far as necessary, preferably until a dry residue isobtained. Where appropriate, the residue is taken up in analcohol/hydrocarbon mixture, especially in methanol/cyclohexane,preferably in the ratios described above, in a relatively low volume.The alcohol phase is evaporated, preferably to dryness, and the alcoholextract is then crystallized from a mixture of an alcohol, especiallyiso-propanol, and a hydrocarbon, especially cyclohexane, preferably inthe ratios described above. The resulting solid crystallized material isthen dissolved in a nitrile/water mixture, preferably as describedabove, especially a 2:3 (v/v) acetonitrile/water mixture, and theresulting feed solution is poured, if required, after splitting in morethan one run, onto a preparative reversed phase column. Elution withnitrile/water, especially as just mentioned, follows. The(aceto-)nitrile of the resulting fractions containing epothilone,especially epothilone A and most especially epothilone B, is removed byevaporation (distillation), and the resulting water phase is extractedwith an ester, especially isopropyl acetate. The ester extract is thenevaporated, preferably to dryness, and subsequently the resultingmaterial is recrystallized, for example an epothilone A fraction iscrystallised directly from an ester/hydrocarbon mixture, e.g. ethylacetate:toluene=2:3, and the epothilone B fraction from an ester,especially ethyl acetate or preferably from an alcohol, especiallymethanol.

Especially with toluene a highly selective eluent (desorbant) has beendiscovered which allows to achieve an approximately 100% yield in halfof the desorption time needed with isopropanol used in WO 99/42602.Surprisingly, the amount of desorbed epothilones is increased, e.g. to130% after desorption with toluene when compared with isopropanoldesorption. (Though this appears prima facie impossible from atheoretical point of view, it nicely illustrates a major advantage ofthe present invention: The result is related to the assay of the loadedresin. As that assay made use of isopropanol for desorption, anincomplete desorption procedure had to be taken as basis leadingoriginally to lower assay values which now turn out to be deceptive).The epothilone mixture can (without or with preceding crystallization)be applied directly to the reverse phase column. The process becomeshighly robust with regard to the solvent amounts, the stirring rates andtemperatures. While for alcohols (e.g. ethanol or isopropanol) understirring a two-phase desorption can be shown where a first amount ofepothilone comprising material is desorbed in a first period of time(explainable possibly by the pore size distribution found in polystyreneXAD-16 which has two maxima of distribution), a second amount after afurther period of time, this undesirable behaviour is not found withtoluene or also dichloromethane where all material is desorbed alreadyin the first period of time. Under comparable conditions, theevaporation residue in the case of isopropanol for epothilones A and Bin one example has been found to be 40 g, with methylene chloride 3.3 g,in the case of toluene only 0.9 g, with e.g. 17-18 g-% of epothilone Bafter desorption from styrene/divinylbenzene copolymer resins obtainedfrom cyclodextrine-comprising culture supernatants as described in WO99/42602, indicating much higher purity. While toluene has the mostsignificant advantages, dichloromethane has as one advantage the ease ofremoval, due to the low boiling point.

The separation of epothilon A and B can also be achieved by performingthe chromatography disclosed herein in its simulated moving bed (SMB)approach. SMB-chromatographies are widely used for the separation ofbinary mixtures, e.g., the separation of racemates on chiral stationaryphases, e.g., the SORBEX processes in the petrochemical industries, likeParex or Molex, or the SAREX process in the sugar industry. Compared tobatch chromatography SMB-chromatography provides the advantage of acontinous countercurrent unit operation which leads to increasedproductivity and reduced mobile phase consumption. Several systematicprocedures for the method development of SMB-chromatographies are knownto the person skilled in the art. Such procedures are described, e.g.,by R.-M. Nicoud, M. Bailly, J. Kinkel, R. M. Devant, T. R. E. Hampe andE. Küsters in Proceedings of the 1^(st) European Meeting on SimulatedMoving Bed Chromatography, (1993), ISBN 2-905-267-21-6, p.65-88; E.Küsters, G. Gerber and F. D. Antia, Chromatographia, 40 (1995) 387; T.Pröll and E. Küsters, J. Chromatogr. A, 800 (1998) 135; or C. Heuer, E.Küsters, T. Plattner and A. Seidel-Morgenstern, J. Chromatogr. A, 827(1998) 175.

The basic parameters for the separation of epothilon A and B with SMBchromatography can be taken directly from the conventional LCseparation. Preferably, reversed phase silica gel (RP 18) is taken asstationary phase and water/acetonitrile-mixtures as mobile phases. Thefinal set of flow rates (for the individual SMB zones and the switchingtime, respectively) can be taken either from a simple flow scheme asdeveloped by E. Küsters, et al in Chromatographia, 40 (1995) 387 orafter careful estimation of adsorption isotherms as laid down in J.Chromatogr. A, 800 (1998) 135 and J. Chromatogr. A, 827 (1998) 175. Thework up of extract and raffinate streams can again be performed asdescribed for the conventional LC separation.

The invention most preferably relates to the processes and methodsdescribed in the subsequent examples.

EXAMPLES

The following Examples serve to illustrate the invention withoutlimiting its scope.

Caution: When handling epothilones, appropriate protective measures mustbe taken, where necessary, in view of their high toxicity.

Example 1 Work-up Procedure for Epothilone B

Desorption of 591.7 kg of charged resin (styrene/divinylbenzenecopolymer resin XAD-16 charged with epothilones A and B from a culturemedium) is effected by stirring the resin in two portions each with 720liters of toluene in four portions for appr. 8 hours. Separation of thetoluene phase from the resin takes place using a suction filter. Thecombined toluene phases are washed in two portions with each 250 l ofwater. After phase separation, the toluene extract is concentrated in a1000 liters reactor to approximately 20-40 liters and afterwardsconcentrated to dryness in a rotary evaporator under vacuum. The resultis a toluene extract of 4.095 kg containing 209 g of epothilone B. Thetoluene extract is dissolved in 16.5 liters of methanol and 24.5 litersof cyclohexane. After addition of 0.8 liters of water phase separationoccurs immediately. The methanol fraction is evaporated to dryness in arotary evaporator under vacuum yielding 1.025 kg evaporation residuecontaining 194 g of epothilone B. The methanol extract is afterwardsbeing crystallized in a solvent mixture consisting of 2.05 litersisopropanol and 10.25 liters cyclohexane, yielding 0.4 kg crystallizedmaterial containing 184 g of epothilone B. The crystals are dissolved in3.2 liters acetonitrile/water=2/3 (v/v) and the resulting feed solutionis transferred in three separate runs onto a preparative reversed phasecolumn (25 kg RP-18 spherical silica gel, YMC-Gel ODS-A 120; 5-15 μm;Waters Corp., Milford, Mass., USA). Elution is effected withacetonitrile/water=2/3 (v/v) as mobile phase with a flow rate of 2.3liters/min; retention time of epothilone A=77-96 min, retention time ofepothilone B 96-119 min. Fractionation is monitored with a UV detectorat 250 nm. The acetonitrile of the combined epothilone B fractions (ofthe three runs) is distilled off and the remaining water phase isextracted with 504 liters of isopropyl acetate. The isopropyl acetateextract is concentrated in a 630 liters reactor to approximately 20-40liters and afterwards concentrated to dryness in a rotary evaporatorunder vacuum. The weight of the evaporation residue of the epothilone Bfractions is 170 g and it has a content of 98.4% according to HPLC(external standard). The resulting material is finally crystallized in2.89 liters of methanol at 0-5° C. yielding 150 g of epothilone B purecrystallisate.

Melting point: 124-125° C.;

¹H-NMR data for epothilone B (500 MHz-NMR, solvent: DMSO-d6. Chemicaldisplacement δ in ppm relatively to TMS. S=singlet, d=doublet,m=multiplet):

δ (Multiplicity) Integral (number of H) 7.34 (s) 1 6.50 (s) 1 5.28 (d) 15.08 (d) 1 4.46 (d) 1 4.08 (m) 1 3.47 (m) 1 3.11 (m) 1 2.83 (dd) 1 2.64(s) 3 2.36 (m) 2 2.09 (s) 3 2.04 (m) 1 1.83 (m) 1 1.61 (m) 1 1.47-1.24(m) 4 1.18 (s) 6 1.13 (m) 2 1.06 (d) 3 0.89 (d + s, overlapping) 6 Σ =41

Example 2 Comparison of Different Desorption Processes for Epothilone BPreparation

360 ml each of an aqueous suspension of the styrene/divinylbenzenecopolymer resin XAD-16 charged with epothilone A and B from a culture ofmyxobacteria by the method described in WO 99/42602 (corresponding to194 g wet Amberlite® XAD-16) are extracted with the solvents and underthe conditions mentioned in the subsequent table in a stirred (commonlab anchor stirrer) glas reactor with a sintered frit at the bottom(home made solid phase batch reactor, inner diameter 10 cm×20 cmlengths, “Stirrer/Frit” hereinafter).

TABLE 1 Comparison of Desorption methods for the manufacture ofEpothilone B Starting resin (resin with absorbed epothilone): #1001theoretical load 70 mg per 360 ml resin suspension (corresponds to 70 mgper 194 g of wet XAD-16) #1003 theoretical load 114 mg per 360 ml resinsuspension (corresponds to 114 mg per 194 g of wet XAD-16)

Variant 1 2 3 4 5 6 Reactor Stirrer/Frit (RT) Stirrer/Frit (RT)Stirrer/Frit (RT) Stirrer/Frit (RT) Reflux (40° C.) Refl. (42-45° C.)Starting Resin #1001 #1001 #1003 #1003 #1003 #1003 Amount of Resin  360 360  360  360  360  180 Suspension (ml) Desorption Solvent IsopropanolMethylenchloride Isopropanol Methylenchloride MethylenchlorideIsopropanol Stirring (rpm)  250  250  250  250  250  250 Volume (l)   8× 0.2 = 1.6   3 × 0.36 = 1.1^(a)   5 × 0.72^(l) = 3.6   3 × 0.72^(h) =2.2   1 × 2.2   1 × 1.8 Desorption time (h)   8   3   5   3   2   2Evaporation (h)   2⁸   0.5   4⁸ None None   2 Water addition (l)   0.7None^(b)   1.15 None None   0.54 Extraction with   1.1 None   2.2 NoneNone   1.1 ethyl acetate (l) Phase separation   24^(c) None   15 NoneNone   15 time (h) Evaporation (h)   2 None   2   1   1   1 Evaporation  5.84^(l)   3.37^(d)   3.31   2.68^(d)   3.24^(l)   1.70 Residue (g)Content (%)   1.26   1.75   2.9   3.7   3.1   3.3 Yield: mg (%)   74(105)^(g)   59 (84)   96 (84)   99 (87)  101 (89)   56 (98) Variant 7 89 10 11 12 Reactor Erl./mag. St.* Stirrer/Frit (RT) Reflux (42-45° C.)Stirrer/Frit (RT) Stir./Frit (RT) Stir./Frit (43° C.) Starting Resin#1003 #1003 #1003 #1003 #1003 #1003 Amount of Resin  100  360  180  360 360  360 Suspension (ml) Desorption Solvent Methylenchl. TolueneIsopropanol Toluene Toluene Toluene Stirring (rpm) ?  640  250  250  640 250 Volume (l)   1 × 0.5 = 0.5   3 × 0.72 = 2.2   1 × 1.8 = 1.8   4 ×0.36 = 1.44   4 × 0.36 = 1.44   4 × 0.36 = 1.44 Desorption time (h)   5  3   6   4   4   4 Evaporation (h) None   6 None None None Wateraddition (l) None   0.5^(l)   0.54   0.5^(k)   0.5^(k)   0.5^(k)Extraction with None None   1.1 None None None ethyl acetate (l) Phaseseparation None   0.1   15   0.1   0.1   0.1 time (h) Evaporation (h)  1   1   1   1   1   1 Evaporation   4.38   0.88   2.32   0.75   0.88  0.95 Residue (g) Content (%)   0.5   17.4   2.69   17.6   17.1   15.8Yield: mg (%)   22 (69)  153 (134)   62.5 (110)  132 (116)^(l)  150(132)  150 (132) ^(a)Not optimized; in case of 2 extractions about 5%yield are lost. ^(b)3-fold back-extraction with water removes no polarcomponent ^(c)First of 3 phase separations overnight ^(d)The material,when compared to ethyl acetate-evaporation residue, can bechromatographed much better on silica gel ^(e)Corresponds to anevaporation residue of about 25 to 30 g ^(f)Drying oven(high vaccum)over the weekend ^(g)Possibly a result of the, concerning the time used,generous first phase separation ^(h)Recovery: 1^(st) extraction = 700ml; 2^(nd) extraction = 660 ml; 3^(rd) extraction = 680 ml ^(i)Recovery:1^(st) extraction = 670 ml; 2^(nd) extraction = 710 ml; 3^(rd)extraction = 720 ml ^(j)The necessity of water addition has not yet beenconfirmed ^(k)Simplifies the removal of the water originallypresent(XAD-16 is covered by water as used) ^(l)In view of the resultsof variant 8, 11 and 12 and the elution curves for all experiments withtoluene, the use of a smaller amount of resin cannot be excluded.,,Erl./mag. St.“stands for magnetic stirring in an Erienmeyer flask.,,Methylenchloride“(,,Methylenchl.“) is dichloromethane,,Stir./Fritt“stands for Stirrer/Frit ,,RT“stands for room temperature

From these experiments and further data, it can be deduced that incomparison with isopropanol the methylenechloride extraction offersbetter selectivity, a shorter time of extraction (approximately by afactor of 2), faster solvent distillation (boiling point of methylenechloride is about 40° C., that of isopropanol 81-83° C.), thetime-intensive and very problematic ethyl acetate/water phase separationis no longer required, the second solvent distillation is no morerequired, so that a lower number of process steps, resulting in lowercontamination risk, better and safer handling, can be used; workup inhalf of the volume (e.g. in 1000 l reactors instead of 2000 l reactors)is possible; the product epothilone B has a better purity profile (fewerby-products with comparable polarity as epothilone B), and theevaporation residue does not sputter and foam as in the case with theethal acetate extraction. For desorption with toluene, a higher yield isobtained in comparison to isopropanol extraction (about 100 instead ofabout 80%), better selectivity can be observed (with isopropanol, aboutthe tenfold amount of by-products is desorbed), the extraction time isshortened considerably (by a factor of about 3), the difficultfiltration after desorption with isopropanol is simplified (theisopropanol extraction was difficult to implement in larger scale), thesecond solvent distillation is no longer required, work-up is possiblein smaller reactors (again, for example, it is possible to use a 1000 linstead of a 2000 l reactor), it is possible to abandon the silica gelchromatography (the evaporation residue after desorption containsalready about 40% of an epothilone A/B-mixture), and the evaporationresidue after desorption does not show the foaming and sputteringobserved for the residue from ethyl acetate extraction.

1. A process for desorbing epothilones from a resin based onstyrene/dinvyl-benzene copolymers charged with epothilones from aculture of myxobacteria sorangium cellulosum, said process making use ofa weakly polar or apolar solvent selected from the group consisting oflower alkyl halogenides and aromatic solvents or a mixture of two ormore such solvents wherein “lower” means that the radical contains uo to7 carbon atoms.
 2. The process according to claim 1 wherein theepothilones to be desorbed are epothilone A or epothilone B.
 3. Theprocess according to claim 1, wherein an aromatic solvent is usedselected from the group consisting of naphthalene, benzene, ornaphthalene or benzene substituted by one or more moieties selected fromthe group consisting of lower alkyl, lower alkoxy, halogen, nitro andlower alkoxy-lower alkyl; wherein the prefix “lower” means that theradical contains up to 7 carbon atoms.
 4. A process according to claim1, further including any further purification steps desirable orrequired to come to the respective epothilones in pure form.
 5. Aprocess according to claim 1 wherein the weakly polar apolar solvent ora mixture of such solvents shows the following characteristics in theeluotropic row determined according to Snyder et al.: X_(e)=0.20-0.40;X_(d)=0.15-0.36; and X_(n)=0.38-0.60 where X_(e) is the proton acceptorparameter, X_(d) is the proton donor parameter and X_(n) is the dipoleparameter, with the proviso that X_(e)+X_(d)+X_(n)=1.
 6. A processaccording to claim 5 wherein the weakly polar or apolar solvent or amixture of such solvents shows the following characteristics in theeluotropic row determined according to Snyder et al.: X_(e)=0.22-0.32;X_(d)=0.17-0.34; and X_(n)=0.39-0.54 where X_(e) is the proton acceptorparameter, X_(d) is the proton donor parameter and X_(n) is the dipoleparameter, with the proviso that X_(e)+X_(d)+X_(n)=1.
 7. A processaccording to claim 1, wherein the epothilones are selected from theepothilones A, B, D, E and F.